PROJECT SUMMARY/ABSTRACT. Tight junctions (TJs) encircle the apical borders of adjacent epithelial cells, and regulate paracellular permeability. The permeability properties of the TJ depend on the expression of transmembrane claudins, which can be cationic or anionic pore-formers or can occlude the paracellular pathway. Critically, TJs must maintain their function in the face of mechanical forces, but how this is accomplished is not well understood. Umbrella cells (UCs) form the outermost layer of the bladder epithelium, and form impermeable TJs, which constitute the urothelial barrier to the pathogens and toxic metabolites in urine. Despite the fundamental importance of the UC TJ in barrier function, and evidence that the UC barrier is disrupted in several diseases of the bladder (e.g., bacterial cystitis, spinal cord injury, and interstitial cystitis), we have limited information about the composition of the UC TJ barrier or the structural and functional changes that allow it to accommodate cycles of filling and voiding. Previous studies from my research group demonstrated that the TJ ring circumscribing each UC doubles in length, and TJ permeability increases when the bladder is filled, and these events are reversed upon voiding. Additionally, claudin expression is altered during the bladder cycle. The mechanisms underlying these changes are unknown. However, I observe that inhibition of exocytosis prevents TJ ring expansion with bladder filling; conversely, inhibition of endocytosis prevents TJ ring contraction upon voiding. I hypothesize that remodeling of the UC TJ depends on changes in the membrane trafficking of claudins, which then modulate TJ permeability. This hypothesis is supported by my preliminary data and will be pursued through two logical aims. In Specific Aim 1, I will determine whether Rab13-dependent exocytosis of pore-forming claudins promotes TJ ring expansion, and increased paracellular permeability during bladder filling. These investigations will employ in vivo biotinylation in combination with exocytosis inhibitors, as well as dominant-active (DA) and dominant-negative (DN) mutants of Rab13, or Rab13-specific shRNAs. Also, I will perform electrophysiological studies to determine if blocking exocytosis prevents the increase in permeability observed with bladder filling. In Specific Aim 2, I will determine whether dynamin-dependent endocytosis of pore-forming claudins leads to TJ ring contraction, and decreased paracellular permeability upon bladder voiding. I will use an in vivo biotinylation internalization assay in conjunction with endocytosis inhibitors and expression of DN-dynamin, to determine if pore-forming claudins are internalized via dynamin-dependent endocytosis. Additionally, I will perform electrophysiological studies to determine if blocking endocytosis, prevents the decrease in permeability observed with bladder voiding. As proposed, this fellowship will provide rigorous training in the field of epithelial cell biology and physiology, which will prepare me for a future career as an independent investigator. Scientifically, the proposed experiments will identify novel pathways of mechanically-regulated TJ dynamics including claudin trafficking to the plasma membrane. Consequently, this proposal will provide a framework to develop therapies targeting pathologies characterized by abnormal claudin expression, and loss of paracellular barrier integrity, such as during cancer metastasis, bacterial cystitis, and interstitial cystitis.